Coinfection Experiments Research Articles

Modification in the ppk gene of Helicobacter pylori during single and multiple experimental murine infections.

The bacterial pathogen Helicobacter pylori is highly adapted to the human stomach, and a high level of polymorphism is observed among clinical isolates. This polymorphism may be the consequence of adaptive changes during colonization, making a strain better able to survive, to evade the immune system, and to provoke a chronic infection. To investigate the mechanisms involved in the acquisition of diversity in H. pylori, mouse models of single infections, coinfections, and superinfections were developed. These experimental infections were conducted with strain SS1, well known to be mouse adapted, and with two strains freshly isolated from infected patients: Hp141 and Hp145. Genetic modifications occurring in these strains were studied over time by comparing randomly selected colonies of the emerging strains to those of the infecting strains by using randomly amplified polymorphic DNA fingerprinting with six different primers and by using PCR to amplify the vacA and cagA genes. We showed that, regardless of the number of infecting strains, only one emerged from the animals and that the establishment of a first strain thwarted the implantation of a second strain. During both a single infection and a coinfection with SS1, Hp141 was replaced by a genetic variant (Hp141v) that overcame SS1 in coinfection experiments. Hp141v exhibited a deletion of a 102-bp repeated sequence within the ppk gene, which encodes polyphosphate kinase (PPK), an enzyme involved in the physiological adaptation of the microbial cell to nutritional and environmental stresses. The deletion led to higher enzymatic activity of PPK, and the variant exhibited a better capacity to colonize mice. Considering that the modified gene is known to be involved in adaptation to a new environment, our results are consistent with an adaptive change in strain Hp141 and suggest that PPK is an important virulence factor in H. pylori.

Pathogen DNA as target for host-generated oxidative stress: role for repair of bacterial DNA damage in Helicobacter pylori colonization.

Helicobacter pylori elicits an oxidative stress during host colonization. This oxidative stress is known to cause lesions in the host DNA. Here we addressed the question as to whether the pathogen DNA is subject to lethal or mutational damage by the host-generated oxidative response. H. pylori Hpnth mutants unable to repair oxidized pyrimidines from the bacterial DNA were generated. H. pylori strains lacking a functional endonuclease III (HpNth) showed elevated spontaneous and induced mutation rates and were more sensitive than the parental strain to killing by exposure to oxidative agents or activated macrophages. Although under laboratory conditions the Hpnth mutant strain grows as well as the wild-type strain, in a mouse infection the stomach bacterial load gradually decreases while the population in the wild-type strain remains stable, showing that endonuclease III deficiency reduces the colonization capacity of the pathogen. In coinfection experiments with a wild-type strain, Hpnth cells are eradicated 15 days postinfection (p.i.) even when inoculated in a 1:9 wild-type:mutant strain ratio, revealing mutagenic lesions that are counterselected under competition conditions. These results show that the host effectively induces lethal and premutagenic oxidative DNA adducts on the H. pylori genome. The possible consequences of these DNA lesions on the adaptability of H. pylori strains to new hosts are discussed.

Type 1 fimbriae and extracellular polysaccharides are preeminent uropathogenic Escherichia coli virulence determinants in the murine urinary tract.

Escherichia coli is the leading cause of urinary tract infections (UTIs). Despite the association of numerous bacterial factors with uropathogenic E. coli (UPEC), few such factors have been proved to be required for UTI in animal models. Previous investigations of urovirulence factors have relied on prior identification of phenotypic characteristics. We used signature-tagged mutagenesis (STM) in an unbiased effort to identify genes that are essential for UPEC survival within the murine urinary tract. A library of 2049 transposon mutants of the prototypic UPEC strain CFT073 was constructed using mini-Tn5km2 carrying 92 unique tags and screened in a murine model of ascending UTI. After initial screening followed by confirmation in co-infection experiments, 19 survival-defective mutants were identified. These mutants were recovered in numbers 101- to 106-fold less than the wild type in the bladder, kidneys or urine or at more than one site. The transposon junctions from each attenuated mutant were sequenced and analysed. Mutations were found in: (i) the type 1 fimbrial operon; (ii) genes involved in the biosyn-thesis of extracellular polysaccharides including group I capsule, group II capsule and enterobacterial common antigen; (iii) genes involved in metabolic pathways; and (iv) genes with unknown function. Five of the genes identified are absent from the genome of the E. coli K-12 strain. Mutations in type 1 fimbrial genes resulted in severely attenuated colonization, even in the case of a mutant with an insertion upstream of the fim operon that affected the rate of fimbrial switching from the 'off' to the 'on' phase. Three mutants had insertions in a new type II capsule biosynthesis locus on a pathogenicity island and were impaired in the production of capsule in vivo. An additional mutant with an insertion in wecE was unable to synthesize enterobacterial common antigen. These results confirm the pre-eminence of type 1 fimbriae, establish the importance of extracellular polysaccharides in the pathogenesis of UTI and identify new urovirulence determinants.

Deletions in the Ac- iap1 gene of the baculovirus Ac MNPV occur spontaneously during serial passage and confer a cell line-specific replication advantage

The PstI-I region of the Autographa californica multicapsid nucleopolyhedrovirus (Ac MNPV) genome was previously shown to be a frequent target of spontaneous deletions during serial virus passage in TN-368 cells (Kumar and Miller, Virus Res. 7 (1987) 335). Analysis of two of these serial passage mutants showed that a portion of the Ac- iap1 gene was deleted. To directly test the effect of loss of Ac- iap1, three different deletions in Ac- iap1 were introduced into recombinant viruses and the ability of these viruses to replicate was examined in two cell lines, TN-368 and SF-21, as well as in two species of insect larvae, Trichoplusia ni and Spodoptera frugiperda. The mutant viruses were indistinguishable from wild type or control revertant virus in their ability to infect larvae of either species. Moreover, no effect was seen on the rate of replication or the overall amounts of budded or occluded virus produced in cultured cells. However, in co-infection experiments using TN-368 cells, it was consistently observed that mutants lacking a functional Ac- iap1 gene out-competed control viruses carrying Ac- iap1. Interestingly, this replication advantage was only evident in the TN-368 cell line, the cell line used for the original serial passage experiments, and not in SF-21 cells.

The structural and functional organization of the yeast mediator complex.

The Mediator complex of Saccharomyces cerevisiae is required for diverse aspects of transcription by RNA polymerase II (pol II). Mediator is composed of two functionally distinct subcomplexes, Rgr1 and Srb4. To identify the structures and functions of each subcomplex, we expressed recombinant proteins for each subunit and assayed their interactions with each other and with basal transcription proteins. The Rgr1 subcomplex is composed of the Gal11 module, which binds activators, and the Med9/10 module. The Med9/10 module is required for both transcriptional activation and repression, and these activities appear to be carried out by two submodules. Proteins in the Med9 submodule interact physically and genetically with Srb10/11, suggesting that the Med9 submodule mediates the repression of pol II. Purified recombinant Srb4 subcomplex stimulated basal transcription of pol II but had little effect on activated transcription and phosphorylation of the C-terminal domain of the Rpb1 subunit of pol II. Both subcomplexes of Mediator interacted with a distinct set of basal transcription factors and pol II. The modular organization of Mediator and the associated functions suggest that the Mediator complex may recruit and/or stabilize the preinitiation complex through several points of contact with transcriptional regulators and basal transcription factors.

Efficient gene activation in cultured mammalian cells mediated by FLP recombinase-expressing recombinant adenovirus.

A recombinant adenovirus (rAd) expressing Cre recombinase derived from bacteriophage P1 has already been extensively used for the conditional gene activation and inactivation strategies in mammalian systems. In this study, we generated AxCAFLP, a rAd expressing FLP recombinase derived from Saccharomyces cerevisiae and carried out quantitative comparisons with Cre-expressing rAd in both in vitro and in cultured cells to provide another efficient gene regulation system in mammalian cells. In the in vitro experiments, the relative recombination efficiency of FLP expressed in 293 cells infected with FLP-expressing rAd was approximately one-thirtieth that of Cre even at 30 degrees C, the optimum temperature for FLP activity, and was approximately one-ninetieth at 37 degrees C. Co-infection experiments in HeLa cells using a target rAd conditionally expressing LacZ under the control of FLP showed that an FLP-expressing rAd, infected at a multiplicity of infection (MOI) of 5, was able to activate the transgene in almost 100% of HeLa cells whereas the Cre-expressing rAd was sufficient at an MOI of 0.2. Since an MOI of 5 is ordinarily used in rAd experiments, these results showed that the FLP-expressing rAd is useful for gene activation strategies and is probably applicable to a sequential gene regulation system in combination with Cre-expressing rAd in mammalian cells.

HtrA homologue of Legionella pneumophila: an indispensable element for intracellular infection of mammalian but not protozoan cells.

Legionella pneumophila replicates within alveolar macrophages, and possibly, alveolar epithelial cells and also within protozoa in the aquatic environment. Here we characterize an L. pneumophila mutant defective in the HtrA/DegP stress-induced protease/chaperone homologue and show that HtrA is indispensable for intracellular replication within mammalian macrophages and alveolar epithelial cells and for intrapulmonary replication in A/J mice. Importantly, amino acid substitutions of two conserved residues in the catalytic domain of (H103mapstoR and S212mapstoA) and in-frame deletions of either or both of the two conserved PDZ domains of HtrA abolish its function. Interestingly, the htrA mutant exhibits a parental-type phenotype in intracellular replication within the protozoan host Acanthamoeba polyphaga. We used a promoterless lacZ fusion to the htrA promoter to probe the phagosomal microenvironment harboring L. pneumophila within macrophages and within A. polyphaga for the exposure to stress stimuli. The data show that expression through the htrA promoter is induced by 12,000- to 20,000-fold throughout the intracellular infection of macrophages but its induction is by 120- to 500-fold within protozoa compared to in vitro expression. Data derived from confocal laser scanning microscopy reveal that in contrast to the parental strain, phagosomes harboring the htrA mutant within U937 macrophages colocalize with the late endosomal-lysosomal marker LAMP-2, similar to killed L. pneumophila. Coinfection experiments examined by confocal laser scanning microscopy show that in communal phagosomes harboring both the parental strain and the htrA mutant, replication of the mutant is not rescued, while replication of a dotA mutant control, which is normally trafficked into a phagolysosome, is rescued by the parental strain. Our data show, for the first time, that the stress response by L. pneumophila (mediated, at least in part, by HtrA) is indispensable for intracellular replication within mammalian but not protozoan cells.

Identification of two Mycobacterium tuberculosis H37Rv ORFs involved in resistance to killing by human macrophages.

The ability of Mycobacterium tuberculosis to survive and replicate in macrophages is crucial for the mycobacterium's ability to infect the host and cause tuberculosis. To identify Mycobacterium tuberculosis genes involved in survival in macrophages, a library of non-pathogenic Mycobacterium smegmatis bacteria, each carrying an individual integrated cosmid containing M. tuberculosis H37Rv genomic DNA, was passed through THP-1 human macrophages three times. Two of the clones recovered from this enrichment process, sur2 and sur3, exhibited significantly increased survival relative to wild-type bacteria. In coinfection experiments, the ratio of sur2 colonies to wild-type colonies was 1:1 at 0 hours but increased to 20:1 at 24 hours post phagocytosis. The ratio of sur3 colonies to wild-type colonies was 1:1 at 0 hours and 5:1 at 24 hours. The M. tuberculosis ORFs responsible for increased survival were shown to be Rv0365c for the sur2 clone and Rv2235 for the sur3 clone. These ORFs encode proteins with as-of-yet unknown functions. We identified two M. tuberculosis ORFs which may be involved in the ability of tubercle bacilli to survive in macrophages.

Enteropathogenicity of Plesiomonas shigelloides and Aeromonas spp. in experimental mono- and coinfection with Cryptosporidium parvum in the intestine of neonatal BALB/c mice

Enteropathogenicity of Plesiomonas shigelloides, Aeromonas hydrophila, A. caviae and A. sobria was studied both in monoinfections and in coinfections with coccidium Cryptosporidium parvum in neonatal BALB/c mice. In monoinfection experiments, neonatal BALB/c mice were orally infected with 7×10 7 or 7×10 8 CFU, respectively, of a strain of P. shigelloides or a strain of an Aeromonas spp. In coinfection experiments, the neonatal mice were, in addition to being orally infected with one of the four bacterial species, orally infected with an inoculum containing 10 5 oocysts of C. parvum. Results from monoinfections with P. shigelloides revealed long-term colonisation of the neonatal mouse intestine by this pathogen, along with associated pathological lesions. The lesions varied in severity from atrophy to necrosis of the mucosal inner surface of the ileum and colon, with predilection to the colon and brush border of colonic enterocytes. The effects of coinfection of P. shigelloides with C. parvum were characterised by bacteremia and heavy colonisation of the intestine by P. shigelloides. In addition, extensive necrotising inflammatory changes in the ileum and colon were accompanied by diarrhoea and deaths of coinfected mice. In contrast, the results from monoinfections of neonatal mice with Aeromonas spp. showed only a short-term colonisation of the intestine by the pathogen. However, when mice were coinfected with A. hydrophila and C. parvum, then the growth of the bacterial species was prolonged, and occurred in both the spleen and intestine. However, no substantial clinical or histopathological changes were observed in mice, whether monoinfected with Aeromonas spp. or coinfected with C. parvum. Our study suggests that experimental monoinfections of neonatal BALB/c mice with P. shigelloides, Aeromonas spp. and C. parvum, together with coinfections (each bacterial species with the protozoan C. parvum), may serve as a useful model to study the initial steps of gastrointestinal colonisation and diarrhoeal disease syndromes caused by enteropathogenic bacteria and protozoa, individually and in combination.

Silencing and reactivation of urease in Yersinia pestis is determined by one G residue at a specific position in the ureD gene.

Yersinia pestis, the plague agent, is a naturally nonureolytic microorganism, while all other Yersinia species display a potent urease activity. In this report we demonstrate that Y. pestis harbors a complete urease locus composed of three structural (ureABC) and four accessory (ureEFGD) genes. Absence of ureolytic activity is due to the presence of one additional G residue in a poly(G) stretch, which introduces a premature stop codon in ureD. The presence of the same additional G in eight other Y. pestis isolates indicates that this mutation is species specific. Spontaneous excision of the extra G occurs at a frequency of 10(-4) to 10(-5) and restores a ureolytic phenotype to Y. pestis. The virulence of two independent ureolytic clones of Y. pestis injected either intravenously, subcutaneously, or intragastrically did not differ from that of the parental strain in the mouse infection model. Coinfection experiments with an equal number of ureolytic and nonureolytic bacteria did not evidence any difference in the ability of the two variants to multiply in vivo and to cause a lethal infection. Altogether our results demonstrate that variation of one extra G residue in ureD determines the ureolytic activity of Y. pestis but does not affect its virulence for mice or its ability to multiply and disseminate.

Carcinoembryonic antigens are targeted by diverse strains of typable and non-typable Haemophilus influenzae.

Haemophilus influenzae (Hi), a commensal of the human respiratory mucosa, is an important cause of localized and systemic infections. We show that distinct strains belonging to typable (THi) and non-typable (NTHi) H. influenzae target human carcinoembryonic antigens (the membrane associated CEA family of cell adhesion molecules, are now termed CEACAMs). All strains of H. influenzae biogroup aegyptius (Hi-aeg) and more than 70% of THi and NTHi strains tested specifically recognize CEACAMI-Fc soluble constructs. Furthermore, transfection of Chinese hamster ovary cells with human CEACAM1 cDNA alone was sufficient for promoting Hi interactions with the transfected cells. The majority of the Hi-aeg strains tested interacted with soluble constructs containing only the N-terminal domain. In contrast, several THi and NTHi strains reacted with soluble constructs only when additional extracellular A and B domains of the receptor were present. The use of monoclonal antibodies confirmed that THi and NTHi strains also interact primarily at the N-domain. We used site-directed mutants of CEACAM1 that contained substitutions at surface exposed amino acids and a molecular model of the N-domain to identify the residues involved in interactions with Hi ligands. The studies show that a common region exposed at the CFG face of the molecule is targeted by diverse Hi strains. However, mutation at distinct sites within this area affected the interactions of distinct strains signifying the potential for tissue tropism via this receptor. Analyses of the molecular basis of interaction with human cell lines and purified CEA show that Hi strains, especially those belonging to Hi-aeg, interact with multiple CEACAMs. Because Neisseria meningitidis (Nm) strains are also known to bind at the CFG face of the receptor, we used Nm and Hi strains in co-infection experiments and demonstrate competition between these mucosal pathogens in colonization of target cells via CEACAMs.

Arabidopsis RTM2 Gene Is Necessary for Specific Restriction of Tobacco Etch Virus and Encodes an Unusual Small Heat Shock–like Protein

Arabidopsis plants have a system to specifically restrict the long-distance movement of tobacco etch potyvirus (TEV) without involving either hypersensitive cell death or systemic acquired resistance. At least two dominant genes, RTM1 and RTM2, are necessary for this restriction. Through a series of coinfection experiments with heterologous viruses, the RTM1/RTM2-mediated restriction was shown to be highly specific for TEV. The RTM2 gene was isolated by a map-based cloning strategy. Isolation of RTM2 was confirmed by transgenic complementation and sequence analysis of wild-type and mutant alleles. The RTM2 gene product is a multidomain protein containing an N-terminal region with high similarity to plant small heat shock proteins (HSPs). Phylogenetic analysis revealed that the RTM2 small HSP-like domain is evolutionarily distinct from each of the five known classes of plant small HSPs. Unlike most other plant genes encoding small HSPs, expression of the RTM2 gene was not induced by high temperature and did not contribute to thermotolerance of seedlings. The RTM2 gene product was also shown to contain a large C-terminal region with multiple repeating sequences.

Helicoverpa armigera Granulovirus Interference with Progression of H. zea Nucleopolyhedrovirus Disease in H. zea Larvae

Capsular proteins from Helicoverpa armigera granulovirus (HaGV) have previously been shown to enhance H. armigera nucleopolyhedrovirus (HaSNPV) infection in H. armigera larvae. Yet, HaGV and HaSNPV, as viable viruses, interfered with one another. In our study, we have examined the effects of co-infection of the slow-killing virus HaGV with the fast-killing virus Helicoverpa zea NPV (HzSNPV) on H. zea larvae. The mortality parameter measured was survival time. Virus stocks had 50% lethal concentrations of 3.2 × 10−9 g HaGV-infected cadavers (GVC) (HaGV) and 32 occlusion bodies (HzSNPV) per cup. Average survival times were 16.8 and 5.5 days for larvae treated with HaGV and HzSNPV, respectively; death of HzSNPV-treated larvae was as early as 72 h posttreatment. In co-infection experiments in which larvae were treated concurrently with both viruses, the viruses competed in typical fashion for host resources. However, interference with disease progression in HzSNPV-fed larvae occurred even when HaGV was fed to larvae up to 36 h after the NPV, a time at which NPV infection should have been well established in host larvae. At death, co-infected larvae were observed microscopically to be filled with HaGV granules rather than HzSNPV polyhedra. The time study results imply that HaGV might be outcompeting HzSNPV by inhibiting its replication. We also observed that H. zea larvae treated with high dosages of HaGV (≥3×10−5 g GVC) were initially stunted but had survival times similar to those of larvae treated with lower dosages.

Effect of the attenuating deletion and of sequence alterations evolving in vivo on simian immunodeficiency virus C8-Nef function.

The simian immunodeficiency virus macC8 (SIVmacC8) variant has been used in a European Community Concerted Action project to study the efficacy and safety of live attenuated SIV vaccines in a large number of macaques. The attenuating deletion in the SIVmacC8 nef-long terminal repeat region encompasses only 12 bp and is "repaired" in a subset of infected animals. It is unknown whether C8-Nef retains some activity. Since it seems important to use only well-characterized deletion mutants in live attenuated vaccine studies, we analyzed the relevance of the deletion, and the duplications and point mutations selected in infected macaques for Nef function in vitro. The deletion, affecting amino acids 143 to 146 (DMYL), resulted in a dramatic decrease in Nef stability and function. The initial 12-bp duplication resulted in efficient Nef expression and an intermediate phenotype in infectivity assays, but it did not significantly restore the ability of Nef to stimulate viral replication and to downmodulate CD4 and class I major histocompatibility complex cell surface expression. The additional substitutions however, which subsequently evolved in vivo, gradually restored these Nef functions. It was noteworthy that coinfection experiments in the T-lymphoid 221 cell line revealed that even SIVmac nef variants carrying the original 12-bp deletion readily outgrew an otherwise isogenic virus containing a 182-bp deletion in the nef gene. Thus, although C8-Nef is unstable and severely impaired in in vitro assays, it maintains some residual activity to stimulate viral replication.

A 1,3-beta-glucanosyltransferase isolated from the cell wall of Aspergillus fumigatus is a homologue of the yeast Bgl2p.

A 1,3-beta-glucanosyltransferase which introduces intrachain 1,6-beta linkages into 1,3-beta-glucan was isolated from the cell wall of Aspergillus fumigatus. The biochemical and molecular characterization of the A. fumigatus transferase showed it was homologous to the Saccharomyces cerevisiae and Candida albicans transferase Bgl2p. A null mutant constructed in A. fumigatus by gene replacement did not show a distinct phenotype from the parental strain. The putative function of this major cell-wall-associated protein is discussed.

Streptococcus pyogenes protein F promotes invasion of HeLa cells.

Although the Gram-positive bacterium Streptococcus pyogenes (group A streptococcus) has been considered an extracellular pathogen which adheres to human mucosal epithelium, the streptococcus possesses invasive capacity for cultured human epithelial cells. This study provides genetic and functional evidence supporting the conclusion that protein F is capable of mediating entry of S. pyogenes into HeLa cells. Using Tn916 insertion mutagenesis or an isogenic S. pyogenes strain with a defined mutation in the gene encoding protein F (prtF), it was observed that the invasive capacity was affected by the levels of surface-exposed protein F, but not by those of M protein. In addition, heterologous expression of protein F on Enterococcus faecalis conferred upon the bacteria an efficient invasive phenotype. Several assays demonstrated that both the fibronectin-binding domains of protein F, UR and RD2, were involved in host-cell invasion. In addition, coinfection experiments of HeLa cells with S. pyogenes and an Escherichia coli K-12 strain expressing an afimbrial adhesin AFA-I showed that the uptake of S. pyogenes did not permit internalization of the E. coli cells.

In vivo analysis of the stability and fitness of variants recovered from foot-and-mouth disease virus quasispecies.

We have analysed the ability to infect pigs of two foot-and-mouth disease virus (FMDV) variants isolated at low frequencies from virus populations (quasispecies) generated in pigs on infection with a parental virus, C-S8c1. A monoclonal antibody-resistant mutant (MARM21), and a variant isolated at early times post-infection (S-3T1), each exhibiting a unique amino acid substitution in VP1, were able to cause disease in pigs, both by direct inoculation or by contact transmission. The symptoms developed were similar to those produced by C-S8c1 or the related virus C-S15c1. The VP1 sequence of viral RNA directly recovered from lesions of infected animals confirmed the stability of the variant genotypes. Pigs infected with S-3T1 consistently showed an advance of 12 to 24 h in the emergence of fever and lesions when compared to animals infected with C-S8c1 or the remaining variants, an observation consistent with its early isolation. The ability of FMDV variants to compete in vivo with C-S8c1 was investigated in co-infection experiments. Analysis of the proportion of each of the competitors in lesions of co-infected pigs revealed that none of the variants was completely overgrown by the parent. However, co-infection with C-S8c1 and MARM21 resulted in lesions in which C-S8c1 was predominant, indicating a selective disadvantage of this variant in swine. In contrast, lesions from swine co-infected with C-S8c1 and S-3T1 contained similar proportions of the two viruses. These results document fitness variations in vivo among components of the mutant spectrum of FMDV quasispecies.

Variations in the interferon-inducing capacity of Sendai virus subpopulations.

Several Sendai virus (SV) preparations, propagated through eggs from the same viral seed, exhibited significantly different capacities to induce interferon (IFN) in human leukocytes (nHu-IFN-alpha). The amount of induced IFN and the numbers of SV IFN-inducing particles (IFP) per cell were determined in dose (SV concentration)-response (IFN yield) curves, kinetics of IFN production, and coinfection experiments with SV preparations that differed in IFN-inducing capacities. The possible role of leukocyte sources and the quality of the SV preparations and of allantoic fluids in affecting the IFN-inducing capacity of SV populations also were tested. The data indicate that different SV preparations induced different amounts of IFN per leukocyte and contained approximately the same concentrations of IFN-inducing particles. There was no apparent correlation between the IFN induced and the apparent quality of the SV preparations examined (EID50, HAU, and EID50/HAU). The leukocyte source and the allantoic impurities of SV preparations did not have any influence on the magnitude of the IFN yield. Similar shapes of the dose-response curves, the absence of any lag in the kinetics of IFN production, and the ability of a viral preparation that induced low yields of IFN to suppress partially a high-yielding inducer suggest that a common mechanism of induction is always present. Hence, propagation of SV in eggs from low multiplicity produced virus stocks that differed significantly in their inducing capacity, suggesting that genetic bottlenecks may be operative.

Identification of artichoke mottled crinkle virus (AMCV) proteins required for virus replication: complementation of AMCV p33 and p92 replication-defective mutants.

Mutagenesis of the artichoke mottled crinkle virus (AMCV) genome and complementation studies between replication-defective mutants were undertaken to identify viral protein(s) essential for AMCV replication. Inoculation of Nicotiana benthamiana protoplasts with mutant transcripts revealed that null mutations in ORFs 1 [tA33(-)], 2 [tA92(-)] and 6 [tA7(-)], as well as an ORF 2 mutation [tA92GED] in the GDD motif of the 92 kDa protein, the putative replicase, prevented accumulation of detectable levels of progeny RNA. Conversely, mutations of ORFs 3 [tA41(-)], 4 [tA21(-)] and 5 [tA19(-)] did not substantially affect the accumulation of AMCV genomic and subgenomic RNAs of both positive and negative polarity. Inoculation of N. benthamiana plants with transcripts impaired in replication revealed that tA92(-) and tA7(-) mutants lead to replicating pseudorevertants. Functional analysis of these pseudorevertants showed that: (i) the double stop codon introduced at the end of ORF 1 to prevent the translational readthrough of the 92 kDa protein reverted to a single amber, ochre or opal codon, giving rise to viable genomes; (ii) the putative 7 kDa protein is not essential for genome viability, although the RNA region spanning ORF 6 plays a role in cis in replication. Finally, the two replication-defective mutants tA33(-) and tA92(-) complemented when co-inoculated to N. benthamiana protoplasts, definitively proving that the 33 kDa protein is essential for tombusvirus genome replication. Analysis of viral RNAs from the coinfection experiments showed that tA92(-) was preferentially amplified over tA33(-).

Bcl-2 expression inSpodoptera Frugiperda Sf-9 andTrichoplusia Ni BTI-Tn-5B1-4 insect cells: Effect on recombinant protein expression and cell viability

The effect of bcl-2 expression on cell viability and recombinant protein synthesis was investigated in the Spodoptera frugiperda Sf-9 and Trichoplusia ni BTI-Tn-5B1-4 (High Fivetrade mark) insect cell lines. It was found that coinfection with a baculovirus expressing bcl-2 [Autographa californica nuclear polyhedrosis virus (AcNPV)-bcl2] extended the life span of High Fivetrade mark cells but not Sf-9 cells when compared to infection with recombinant baculoviruses expressing either human tissue plasminogen activator (AcNPV-tPA) or Escherichia coli beta-galactosidase (AcNPV-betagal). Similar results were obtained in coinfection experiments; i.e., AcNPV-bcl2 coinfection increased the life span of High Fivetrade mark cells over that of cells infected with either AcNPV-tPA or AcNPV-betagal alone, but they did not affect the life span of coinfected Sf-9 cells. Coinfection of Sf-9 cells with AcNPV-bcl2 and AcNPV-betagal resulted in a decrease in the maximum beta-gal expression levels of over 90% when compared to infection with AcNPV-betagal alone. A similar trend was found in the beta-gal mRNA levels. Coinfection also resulted in a reduced beta-gal expression level in High Fivetrade mark cells, but the reduction was consistent with what would be expected when two recombinant viruses compete for use of the cellular machinery. In contrast to the inhibitory effect of AcNPV-bcl2 coinfection on betagal expression, t-PA expression levels were either not affected (Sf-9 cells) or were increased 50% (High Fivetrade mark cells) over those obtained by infection with AcNPV-tPA alone. These results support the hypotheses that bcl-2 can inhibit transcription of genes under polyhedrin promoter control and that beta-gal expression levels, but not t-PA expression levels, are controlled at the transcriptional level. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 380-390, 1997.